An interaction between the mammalian DNA repair protein XRCC1 and DNA ligase III.

XRCC1, the human gene that fully corrects the Chinese hamster ovary DNA repair mutant EM9, encodes a protein involved in the rejoining of DNA single-strand breaks that arise following treatment with alkylating agents or ionizing radiation. In this study, a cDNA minigene encoding oligohistidine-tagged XRCC1 was constructed to facilitate affinity purification of the recombinant protein. This construct, designated pcD2EHX, fully corrected the EM9 phenotype of high sister chromatid exchange, indicating that the histidine tag was not detrimental to XRCC1 activity. Affinity chromatography of extract from EM9 cells transfected with pcD2EHX resulted in the copurification of histidine-tagged XRCC1 and DNA ligase III activity. Neither XRCC1 or DNA ligase III activity was purified during affinity chromatography of extract from EM9 cells transfected with pcD2EX, a cDNA minigene that encodes untagged XRCC1, or extract from wild-type AA8 or untransfected EM9 cells. The copurification of DNA ligase III activity with histidine-tagged XRCC1 suggests that the two proteins are present in the cell as a complex. Furthermore, DNA ligase III activity was present at lower levels in EM9 cells than in AA8 cells and was returned to normal levels in EM9 cells transfected with pcD2EHX or pcD2EX. These findings indicate that XRCC1 is required for normal levels of DNA ligase III activity, and they implicate a major role for this DNA ligase in DNA base excision repair in mammalian cells.

Induction of mutations by chemical agents at the hypoxanthine-guanine phosphoribosyl transferase locus in human epithelial teratoma cells.

Induction of 6-thioguanine (TG) resistance by chemical mutagens was examined in a line of cells derived from a human epithelial teratocarcinoma cell clone. The cells, designated as P3 cells, have a stable diploid karyotype with 46(XX) chromosomes, including a translocation between chromosomes 15 and 20. Efficient recovery of TG-resistant mutants induced by the direct-acting mutagens: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG); 7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10 -tetrahydrobenzo[a]pyrene (BPDE); and benzo[a]pyrene (B[a]P); activated in a cell-mediated assay, required an expression time of 7 days and a saturation density of 2 X 10(4) cells/60-mm petri dish. The TG-resistant mutant cells induced by MNNG and BPDE maintained their resistant phenotype 4-6 weeks after isolation. This mutant phenotype was associated with a more than 10-fold reduction in hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity relative to that of the parental P3 cell line, which was shown to catalyze the formation of 4.6 pmoles inosine-5'-monophosphate (IMP)/min/microgram protein. Induction of TG resistance was also observed in P3 cells cocultivated in a cell-mediated assay with human breast carcinoma cells, which are capable of polycyclic aromatic hydrocarbon (PAH) metabolism, after treatment with the carcinogenic PAHs: B[a]P, chrysene, 7,12-dimethylbenz[a]anthracene (DMBA), and 3-methylcholanthrene (MCA). The degree of mutant induction in this assay was related to the carcinogenic potency of these PAHs in experimental animals. The most potent mutagen was DMBA, followed in decreasing order by MCA, B[a]P, and chrysene. DMBA, at 0.4 microM, increased the frequency of mutants for TG resistance from 2 for the control to about 200 TG-resistant mutants/10(6) colony-forming cells (CFC). Benzo[e]pyrene (B[e]P) and pyrene, which are not carcinogenic, were not effective in the assay. None of the PAHs was mutagenic in the P3 cells cultivated in the absence of the PAH-metabolizing cells. These results indicate that the P3 cells can be useful for the study of mutagenesis at the HGPRT locus by direct-acting chemical mutagens, as well as by chemicals activated in a cell-mediated assay.

Keratinocyte cell-mediated mutagenesis assay: correlation with in vivo tumor studies.

A murine keratinocyte cell-mediated mutagenesis assay was characterized and examined as an in vitro model system for studying the biotransformation of promutagens/procarcinogens by mouse skin. The assay used living cultured newborn SENCAR keratinocytes for the metabolic activation of promutagens and Chinese hamster lung V-79 fibroblasts for detection of resulting mutagens. Mutations at, or affecting, the hypoxanthine-guanine phosphoribosyltransferase locus were scored by resistance to 6-thioguanine. The relative mutagenicities of several polycyclic aromatic hydrocarbons (PAHs) in the cell-mediated assay correlated with the in vivo skin tumorigenicity of the PAHs determined in a two-stage carcinogenesis protocol. Metabolic activation of the promutagenic PAHs to ultimate mutagens was dependent upon the presence of the cultured keratinocyte feeder layer. 7,8-Benzoflavone, a potent inhibitor of 7,12-dimethylbenz[a]anthracene (DMBA)-dependent initiation in mouse skin, inhibited DMBA-dependent mutagenesis in the cell-mediated assay in a concentration responsive manner. The non-PAH promutagens, dimethylnitrosamine (DMN) and sterigmatocystin (STC) were both activated by cultured keratinocytes to cytotoxic derivatives. DMN was neither mutagenic in the cell-mediated assay nor tumorigenic in mouse skin when tested in a two-stage carcinogenesis protocol. STC was weakly mutagenic and tumorigenic in mouse skin.

Cell-mediated mutagenicity in Chinese hamster V79 cells of dibenzopyrenes and their bay-region fluorine-substituted derivatives.

The polycyclic aromatic hydrocarbons dibenzo(a,i)pyrene and dibenzo(a,h)pyrene, each of which possesses two bay regions, and their bay-region difluorinated derivatives were tested for mutagenicity for ouabain and 6-thioguanine resistance in Chinese hamster V79 cells. Since V79 cells do not metabolize polycyclic aromatic hydrocarbons, mutagenesis was tested in both the presence and the absence of golden hamster embryo cells capable of metabolizing polycyclic aromatic hydrocarbons. Neither of the dibenzopyrenes nor their fluorinated derivatives were mutagenic in the absence of the golden hamster embryo cells. In the presence of these cells (cell-mediated assay), both dibenzopyrenes were mutagenic, whereas the difluorinated derivatives, 2,10-difluorodibenzo-(a,i)pyrene and 3,10-difluorodibenzo(a,h)pyrene, either were inactive or exhibited (on a dose basis) a weak response. However, the mutagenicity of the dibenzopyrenes was eliminated when they were coincubated with 7,8-benzoflavone, a mixed-function oxidase inhibitor. The results suggest that metabolic oxidation of these polycyclic aromatic hydrocarbons at the bay region (presumably to diol-epoxides) is required for a mutagenic response in the cell-mediated assay.

Mutagen-induced resistance to mycophenolic acid in hamster cells can be associated with increased inosine 5'-phosphate dehydrogenase activity.

Cell variants resistant to the cytotoxic effect of mycophenolic acid, an inhibitor of IMP dehydrogenase (IMP:NAD+ oxidoreductase, EC 1.2.1.14), were selected by a one-step procedure from Chinese hamster V79 cells. The frequency of these variants was increased in a dose-dependent manner after treatment with the mutagen N-methyl-N'-nitro-N-nitrosoguanidine and after an expression time of 8 days. The degree of resistance in five of the six isolated cell variants was associated with a comparable increase in the specific activity of IMP dehydrogenase, which was 3- to 6-fold higher than that of the parent V79 cells. The IMP dehydrogenase activity from both the variants and the V79 cells had a similar affinity for the substrate IMP with a Km of about 20 microM and a similar response to mycophenolic acid with a Ki of 12-16 nM. It is suggested that cell variants with an altered regulation of IMP dehydrogenase activity may be helpful in studying the control of nucleic acid biosynthesis, cell growth, and carcinogenesis. Mycophenolic acid resistance also may be useful as a marker in short-term assays for the identification of potential chemical carcinogens.